Figure 6.
Immunomodulatory Effects of EVs from MSCs with Manipulation of TGF-β1 and PTX3 Expression
(A) IFN-γ ELISA with conditioned medium of anti-CD3/28-stimulated splenocytes for 72 h with or without recombinant human (rh) proteins (0–200 ng/mL) (n = 4). (B) ELISAs for protein levels of TGF-β1, TGFβI, PTX3, and LUM in MSC-EVs derived from MSCs transfected with target siRNAs. (C) IL-2 and IFN-γ ELISAs with conditioned medium of splenocytes activated by plate-bound anti-CD3 for 24 h with EVs derived from MSCs transfected with target siRNAs (n = 4). (D and E) Ocular surface photography with lissamine green staining (D) and quantitation of corneal epithelial defects using a standardized scoring system (E). (F) Aqueous tear production as quantified by a phenol red thread test and numeration of conjunctival goblet cells on PAS-stained conjunctival specimens. A dot indicates data from a single animal. (G) IFN-γ ELISA with conditioned medium of splenocytes activated by plate-bound anti-CD3 for 24 h. (H) IL-6 ELISA with conditioned medium of LPS-stimulated splenocytes for 24 h with EVs derived from PD15 MSCs transfected with target DNA plasmids (n = 4). All data are presented as means ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by one-way ANOVA with Dunnett’s or Tukey’s test. (I) Western blot assays with nuclear factions (NFs) of anti-CD3/28-stimulated splenocytes for 3 h with or without MSC-EVs (3 × 109 particles/mL). (J) Western blot assays with total cell lysates (TCLs) of anti-CD3/28-stimulated splenocytes for 30 min with or without MSC-EVs (3 × 109 particles/mL). (K) Western blot assays with nuclear factions (NFs) of LPS-stimulated splenocytes for 1 h with or without MSC-EVs (3 × 109 particles/mL).